The transfected cells were lapped with RIPA lysis and extraction buffer (Sangon Biotech, Shanghai, China). The protein concentration was detected using a BCA protein assay kit (Pierce, U.S.A.). The protein (20 μg) was subjected to a 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Sigma, U.S.A.). Then the membranes were incubated with primary antibodies (matrix metallopeptidase 2 (MMP2), MMP9, c-Jun N-terminal kinase (JNK), p-JNK, c-Jun, p-c-Jun; 1:1000 dilution) at 4°C overnight, followed by incubation with horseradish peroxidase labeled secondary antibody (1:1000 dilution) for 1 h at room temperature. The bands were visualized using electrochemiluminescence (ECL) chromogenic substrate. The intensity of the bands was quantified by densitometry. GAPDH was served as the internal control.
Ripa lysis and extraction buffer
Manufactured by Sangon
Sourced in China
RIPA lysis and extraction buffer is a solution used for cell lysis and protein extraction. It contains a combination of detergents and salts to solubilize cellular components and release proteins from cells.
Automatically generated - may contain errors
2 protocols using ripa lysis and extraction buffer
1
Quantifying Protein Expression Dynamics
2
Spinal Cord Injury Protein Analysis
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Injured spinal cords were cut into pieces and treated with trypsin. The proteins were extracted using RIPA lysis and extraction buffer (Sangon Biotech, China) and their concentrations were measured using a Thermo Scientific microBCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The proteins were separated on 10% SDS-PAGE and transferred onto Millipore PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with silk and incubated at 4ºC for 12 h with primary antibodies against bFGF (1: 500 dilution, Millipore), EGF (1: 500 dilution, Millipore), and GAPDH (1: 1,000 dilution, Biogenesis, Bournemouth, UK). After washing, the membranes were incubated with HRP-conjugated secondary antibodies, and immunoreactive bands were analyzed using an ECL kit (Amersham Biosciences, Little Chalfont, UK).
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